Germinal centers determine the prognostic relevance of tertiary lymphoid structures and are impaired by corticosteroids in lung squamous cell carcinoma

In solid tumors, the presence of lymph node-like structures called tertiary lymphoid structures (TLS) is associated with improved patient survival. However, little is known about how TLS form in cancer, how their function affects survival, and whether they are affected by cancer therapy. In this study, we used multi-spectral microscopy, quantitative pathology and gene expression profiling to analyze TLS formation in human lung squamous cell carcinoma (LSCC) and in an experimental model of lung TLS induction. We identified a niche of CXCL13+ perivascular and CXCL12+LTB+ and PD-L1+ epithelial cells supporting TLS formation. We also characterized sequential stages of TLS maturation in LSCC culminating in the formation of germinal centers (GC). In untreated patients, TLS density was the strongest independent prognostic marker. Further, TLS density correlated with GC formation and expression of adaptive immune response-related genes. In patients treated with neoadjuvant chemotherapy, TLS density was similar but GC formation was impaired and the prognostic value of TLS density was lost. Corticosteroids are co-administered with chemotherapy to manage side effects in LSCC patients, so we evaluated whether they impaired TLS development independently of chemotherapy. TLS density and GC formation were each reduced in chemotherapy-naive LSCC patients treated with corticosteroids before surgery, compared to untreated patients, a finding that we confirmed in the experimental model of lung TLS induction. Overall, our results highlight the importance of GC formation in TLS during tumor development and treatment

Le virus SARS-CoV-2 utilise différentes voies d’entrée pour infecter les cellules

International audienceNo abstract availabl

Entrée cellulaire des phlébovirus chez l’hôte mammifère

National audiencePhleboviruses constitute a large group of arthropod-borne viruses (arboviruses), mainly transmitted to their hosts by sandflies and ticks, occasionally by mosquitoes. These viruses have a worldwide distribution and many cause serious diseases - often fatal - in both domestic animals and humans. The global warming, the apparent wide distribution of arthropod reservoirs, and the increasing number of outbreaks show that phleboviruses must be taken seriously as emerging disease agents. This review proposes to focus on the early steps of phlebovirus infection, from virus binding to penetration into the cytosol. We address the most recent knowledge and advances in the entry of these viruses into vertebrate host cells, including virus receptors, cellular factors, endocytic pathways, and fusion

Zika virus prM protein contains cholesterol binding motifs required for virus entry and assembly - Molecular Dynamics Simulation Dataset

<p>The molecular dynamics (MD) simulation dataset. The contents:</p> <ul> <li><strong>5ire_BIOMT_expanded.pdb</strong>: The complete biological assembly of the cryo-EM structure of Zika Virus (PDB ID:5IRE) </li> <li><strong>5ire_Mprotein_BIOMT_expanded.pdb</strong>: The M proteins extracted from the complete biological assembly of the cryo-EM structure of Zika Virus (PDB ID:5IRE).  The biological assembly shows the dimeric organization of M proteins.</li> <li><strong>0chol.zip, 10chol.zip, 20chol.zip, and 30chol.zip</strong> contain simulation input and output files for the simulated membrane compositions: 0:100, 10:90, 20:80, 30:70 (mol%:mol%) Cholesterol:POPC, respectively.  <ul> <li>In each zip file, there are 5 directories: <strong>wt, R253L+F257A, R253L+F257S, K275L+Y278A, K275L+Y278S</strong> corresponding to each simulated M protein dimer variant: wild type, CARC 2-A, CARC 2-S, CARC 3-A, and CARC 3-S. In each directory, there are the following files: <ul> <li><strong>toppar</strong>: This directory contains all force field topologies and parameters</li> <li><strong>topol.top</strong>: GROMACS topology (top) file</li> <li><strong>index.ndx</strong>: GROMACS index (ndx) file</li> <li><strong>prod.mdp</strong>: GROMACS MD parameters (mdp)  file</li> <li><strong>0, 1, 2, 3, 4, 5, 6, 7, 8, 9</strong>: These directories contain the simulation inputs and outputs for each simulation repeat. In each of these directories, there are the following files:  <ul> <li><strong>t0.pdb</strong>: The pdb file of the starting coordinates</li> <li><strong>prod0.tpr</strong>: GROMACS binary run input (tpr) file </li> <li><strong>prod0.edr</strong>: GROMACS energy (edr) file</li> <li><strong>prod0.gro</strong>: GROMACS output coordinates and velocities after 1 microsecond of simulation</li> <li><strong>prod0.cpt</strong>: GROMACS checkpoint file after 1 microsecond of simulation</li> <li><strong>noW.pdb</strong>: The pdb file of the starting coordinates with all water molecules removed</li> <li><strong>noW.xtc</strong>:  GROMACS compressed trajectory (xtc) file with all water molecules removed</li> </ul> </li> </ul> </li> </ul> </li> </ul&gt

Novel toscana virus reverse genetics system establishes NSS as an antagonist of type I interferon responses

The sand fly-borne Toscana virus (TOSV) is the major cause of human meningoencephalitis in the Mediterranean basin during the summer season. In this work, we have developed a T7 RNA polymerase-driven reverse genetics system to recover infectious particles of a lineage B strain of TOSV. The viral protein pattern and growth properties of the rescued virus (rTOSV) were found to be similar to those of the corresponding wild-type (wt) virus. Using this system, we genetically engineered a TOSV mutant lacking expression of the non-structural protein NSs (rTOSVϕNSs). Unlike rTOSV and the wt virus, rTOSVϕNSs was unable to (i) suppress interferon (IFN)-b messenger RNA induction; and (ii) grow efficiently in cells producing IFN-b. Together, our results highlight the importance of NSs for TOSV in evading the IFN response and provide a comprehensive toolbox to investigate the TOSV life cycle in mammalian and insect host cells, including several novel polyclonal antibodies.</p

The phenuivirus Toscana virus makes an atypical use of vacuolar acidity to enter host cells.

Toscana virus is a major cause of arboviral disease in humans in the Mediterranean basin during summer. However, early virus-host cell interactions and entry mechanisms remain poorly characterized. Investigating iPSC-derived human neurons and cell lines, we found that virus binding to the cell surface was specific, and 50% of bound virions were endocytosed within 10 min. Virions entered Rab5a+ early endosomes and, subsequently, Rab7a+ and LAMP-1+ late endosomal compartments. Penetration required intact late endosomes and occurred within 30 min following internalization. Virus entry relied on vacuolar acidification, with an optimal pH for viral membrane fusion at pH 5.5. The pH threshold increased to 5.8 with longer pre-exposure of virions to the slightly acidic pH in early endosomes. Strikingly, the particles remained infectious after entering late endosomes with a pH below the fusion threshold. Overall, our study establishes Toscana virus as a late-penetrating virus and reveals an atypical use of vacuolar acidity by this virus to enter host cells

Quantitative proteomics of Uukuniemi virus - host cell interactions reveals GBF1 as proviral host factor for phleboviruses.

Novel tick-borne phleboviruses in the Phenuiviridae family, which are highly pathogenic in humans and all closely related to Uukuniemi virus (UUKV), have recently emerged on different continents. How phleboviruses assemble, bud, and exit cells remains largely elusive. Here, we performed high-resolution, label-free mass spectrometry analysis of UUKV immuno-precipitated from cell lysates and identified 39 cellular partners interacting with the viral envelope glycoproteins. The importance of these host factors for UUKV infection was validated by silencing each host factor by RNA interference. This revealed Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1 (GBF1), a guanine nucleotide exchange factor resident in the Golgi, as a critical host factor required for the UUKV life cycle. An inhibitor of GBF1, Golgicide A, confirmed the role of the cellular factor in UUKV infection. We could pinpoint the GBF1 requirement to UUKV replication and particle assembly. When the investigation was extended to viruses from various positive and negative RNA viral families, we found that not only phleboviruses rely on GBF1 for infection, but also Flavi-, Corona-, Rhabdo-, and Togaviridae In contrast, silencing or blocking GBF1 did not abrogate infection by the human adenovirus serotype 5 and immunodeficiency retrovirus type 1, the replication of both occurs in the nucleus. Together our results indicate that UUKV relies on GBF1 for viral replication, assembly and egress. This study also highlights the proviral activity of GBF1 in the infection by a broad range of important zoonotic RNA viruses

Entry of Toscana virus (TOSV) into cells.

The time and pH scale on the right side illustrates the typical acidity and timing of cellular cargoes to reach the respective endosomal compartments. +/++ indicate the amount of Rab7a and LAMP-1.</p

Toscana virus (TOSV) entry into iPSC-derived human neurons depends on endosomal acidification.

(A to C) iPSC-derived neurons (A) and A549 cells (B and C) were pretreated with agents that elevate endosomal pH at the indicated concentrations and were infected with TOSV at MOI 10 for 8 h and MOI 2 for 6 h, respectively, in the continuous presence of ammonium chloride (NH4Cl), chloroquine, bafilomycin A1, and concanamycin B. Infection was analyzed by flow cytometry, and data were normalized to those of control samples without inhibitor treatment.</p

This Excel workbook includes all the numerical values used to produce the graphs shown in this study.

This Excel workbook includes all the numerical values used to produce the graphs shown in this study.</p